Causes of self-induced limitation of glyphosate translocation were studied in sugar beet plants that were susceptible to or tolerant of (Roundup Ready) glyphosate. Glyphosate was taken up in both types of plants at the same rate and essentially stopped at the same time, around 4 h after [14C]glyphosate was applied in 1% Roundup formulation. Tolerant plants continued to export glyphosate at a significant rate during the entire 30-h observation period while susceptible plants stopped after only 10 h and exported only half the amount. Herbicide was applied under three different experimental protocols to help identify causes for the inhibition of carbon and glyphosate translocation. Comparing the effect of 1% Roundup applied to source leaves of susceptible plants, during either the first half of the day or the end of the day, showed that glyphosate acting directly on source leaves under high light essentially stopped translocation. Glyphosate nearly completely inhibited photosynthesis and carbon export during the remaining 9 h of the light period but produced no observable effects on photosynthesis, C3-cycle carbon metabolism, or translocation in tolerant plants. Inhibition of carbon translocation and glyphosate translocation coincided, confirming that glyphosate export was inhibited by disruption of a process that drives carbon export. Applying glyphosate to all source leaves, except the two in which photosynthesis and export were being measured, revealed the effects of sink tissue inhibition on source leaf processes in the absence of direct effects on the source leaves. Import into the monitored sink leaf was reduced by 50% after about 3 h but the inhibition of import had a relatively small effect on carbon export and photosynthesis in the two source leaves not directly exposed to the herbicide. The results show that the glyphosate-induced disruption of C3 cycle metabolism under high light was a key factor in the marked inhibition of photosynthesis and the rapid halting of carbon and glyphosate translocation in sugar beets.